Actinomycin D is used for treatment of paediatric cancers; however, a large inter-patient pharmacokinetic (PK) variability and hepatotoxicity are significant limitations to its use and warrant further investigation. Elimination of actinomycin D may be mediated by transporters, as the drug does not seem to undergo significant metabolism. We investigated the role of solute carrier (SLC) transporters in actinomycin D PK. Fourteen key SLCs were screened through probe substrate uptake inhibition by actinomycin D in HEK293 cells. Uptake of actinomycin D was further studied in candidate SLCs by measuring intracellular actinomycin D using a validated LCMS assay. Pharmacogenetic analysis was conducted for 60 patients (Clinical trial: NCT00900354), who were genotyped for SNPs for OAT4 and PEPT2. OAT4, OCT2, OCT3 and PEPT2 showed significantly lower probe substrate uptake (mean±SD 75.0±3.5% (p < 0.0001), 74.8±11.2% (p = 0.001), 81.2±14.0% (p = 0.0083) and 70.7±5.7% (p = 0.0188)) compared to that of control. Intracellular accumulation of actinomycin D was greater compared to vector control in OAT4-transfected cells by 1.5- and 1.4-fold at 10min (p = 0.01) and 20min (p = 0.03), and in PEPT2-transfected cells by 1.5- and 1.7-fold at 10min (p = 0.047) and 20min (p = 0.043), respectively. Subsequent clinical study did not find a significant association between OAT4 rs11231809 and PEPT2 rs2257212 genotypes, and actinomycin D PK parameters such as clearance (CL) and volume of distribution (Vd). Transport of actinomycin D was mediated by OAT4 and PEPT2 in vitro. There was a lack of clinical significance of OAT4 and PEPT2 genotypes as predictors of actinomycin D disposition in paediatric cancer patients.